Targeted Next-Generation Sequencing for Diagnostics and Forensics.

Abstract

Pathogen-specific, targeted assays such as real-time PCR are generally used for clinical diagnosis of infectious and biothreat diseases as highlighted in the recent West African Ebola virus outbreak where real-time PCR was the primary diagnostic for critical treatment decisions. However, current diagnostic algorithms rarely require determination of a single etiologic agent outside the context of an outbreak response but rather involve interrogation across a spectrum of pathogens with similar pathology and/or epidemiology. The necessity for multiple pathogen interrogation exposes the critical gap in availability of higher-multiplex diagnostics. This caveat is exacerbated with biothreat agents where initial symptoms of an infection are often similar to a myriad of less consequential pathogens. Development of diagnostic platforms is a daunting challenge given the plethora of human pathogens spanning both infectious and biothreat diseases. In general, infectious disease diagnostics must meet defined metrics for performance in clinical matrices as well as show validated efficacy for use in an actionable context to impact patient care. Detection of virulence markers to discriminate microbiota from pathogenic events and the ability to detect the spectrum of pathogen heterogeneity are critical aspects to an ideal diagnostic platform. Biothreat diagnostics include all of these requirements in addition to the need for microbial forensics/attribution. The detection of a biothreat incident (e.g., Bacillus anthracis ), environmental or clinical, has substantial consequences including public angst as well as mobilization of military, public health, and law enforcement responses, the latter of these responses requiring forensic attribution. Detection of both clinical and environmental biothreat incidents remains a perennial issue that requires some level of preparedness to mitigate situations proactively. Next-generation sequencing (NGS)2 offers a potential solution to challenges for both diagnostic and environmental detection of infectious disease and biothreats. Specifically, NGS has the potential to sequence all nucleic acids within a sample, allowing for near …